Mayfield_BSL2_Manual_2023_Nov_MostCurrent

Mayfield_BSL2_Manual_2023_Nov_MostCurrent.pdf

General Laboratory Practices for BSL-2 Laboratories

1. Dayne Mayfield Lab

A general template for this document can be found here.  The principal investigator is responsible for including laboratory and protocol specific procedures for addressing hazards in their laboratory.  This plan must be adopted by the Institutional Biosafety Committee (IBC).

The official “University of Texas Laboratory Safety Manual” is located on the top shelf above the desk in MBB 1.124.  Please refer to it if you require a more in-depth description of university safety responsibilities, emergency procedures, fundamentals of laboratory safety, and basic and/or specific procedures for working with chemicals and biological hazards

Additionally, the EHS website is has the most up-to-date information. Always consult it if you have any questions/concerns.

This BSL2 manual must be reviewed and signed annually by all lab members.

I have read, understand, and agree to adhere to the biosafety procedures contained within:

Printed Name              Signature                     Date

PI:

_Mayfield, R. Dayne_               

Written (Date):  _______________

Last Revision (Date):  __2/23/23__

Reviewed by BSO (Date):  ____________

Adopted by the IBC (Date10/19/2022): 

Table of Contents

Principal Investigator Responsibilities………………………………………...…    3

Laboratory Staff/Student Responsibilities…………………………………….....    4

General Emergency Information……………………………………….………...    4

Emergency Contacts……………………………………….…………………….     4

Fire Alarms/Extinguishers……………………………...……….………………..    5

Eyewashes……………………………………….……………………………….    5

Spills in the Laboratory……………………………………….………………….    5

Biological Spill……………………………………….…………………………..    6

Spills Outside the Biosafety Cabinet……………………………………….…….    6

Spills Inside the Biosafety Cabinet…………………………………………..…..    6

Spills Inside Equipment……………………………………….………………….   6

Chemical Spill……………………………………….……………………………   6

Radiation Spill ……………………………………….………………...…………   7

Exposures in the Laboratory……………………………………….…………….     7

      Biological Exposure……………………………………….……………………..    7

Chemical Exposure……………………………………….……………………...     7

      Radiation Exposure……………………………………….……………………...    8

BSL-2 Safety Procedures……………………………………….………………..    8

New Employees……………………………………….…………………………     9

Training……………………………………….………………………………….    9

Medical Surveillance……………………………….…………………………….    11

PPE……………………………………….………………………………………    11

Biohazard Warning Signs and Posting……………………………………….…..    12

Biological Safety Cabinets (BSC’s) ……………………………………….…….    12

Biological Waste Disposal……………………………………….………………    13

Chemical Safety……………………………………………………………….…    13

Fume Hood Use……………………………………….…………………………     14

Housekeeping………………………………………………………………….…    14

Laboratory Close Outs/Equipment Disposal…………………………………….     14

Security……………………………………….………………………………….     14

Controlled substances……………………………………………………………     15

Select Agents/Toxins………………………………………………………….…     16

Sharps……………………………………….……………………………………    16

Shipping Biological Material……………………………………….……………    17

Transporting Biological Material……………………………………….………..    17

ClusterMarket…………………………………………………………………….    17

Useful Web Sites……………………………………….………………………...    17

Appendix A……………………………………….…………..………………….    18

Appendix B……………………………………….………………………………    20

Appendix C……………………………………….………………………………    21

Principal Investigator Responsibilities

The Principal Investigator (PI) has the primary responsibility for ensuring that their laboratory is safe.  They must adhere to all guidelines and regulations.  They are responsible for the safe use of biological, chemical and radioactive materials in their laboratory.

In addition, the PI must:

            Limit personnel, student, and visitor exposure to hazards to the lowest practical level.

            Provide special safety considerations for individuals under the age of 18

            Apply the recommended biosafety level for the work being conducted

            Be familiar with the required medical surveillance for each type of agent in their laboratory and ensure staff/students/visitors have medical clearance

            Develop written lab specific safety procedures and train their personnel on them 

            Maintain documentation of training

            Provide PPE and instruction on use

            Ensure waste is properly disposed

            Report spills, exposures or incidents to Environmental Health & Safety (EHS)

            Conduct periodic drills of emergency procedures

Laboratory Staff/Student Responsibilities 

            Know the biological materials and procedures used in the laboratory

            Follow approved lab procedures and safety guidelines

            Know emergency procedures

            Complete all required training before conducting any lab activity

            Complete recurring trainings by the due-date

            Report any unsafe conditions to the PI, EHS or the RRT

            Utilize all required Personal Protective Equipment (PPE)

            Use appropriate lab equipment and containment facilities

General Emergency Information

Emergency Contacts

If there is an emergency, call 911 to reach UTPD.  UTPD will then summon assistance from the City of Austin Emergency Services.  If there is a fire or explosion that you cannot control, evacuate the area immediately.  Pull the fire alarm and then call 911 from a safe location.  For other emergencies, dial 911 for UTPD.

If any emergency or significant spill/exposure occurs in the laboratory, immediately notify EHS and your lab supervisor/PI. ALL lab incidents (spills, injuries, near-misses) should be reported to EHS using their online form. Phone support (24hr) is also available at 512-471-3511.

Fire Alarms/Extinguishers

Locations of fire pull station alarms:

(1)  Exterior entrance – south wing of first floor

(2)  South end of first floor hallway

(3)  Loading dock exterior door

Location of fire extinguishers:

(1)  MBB 1.124 next to telephone

(2)  MBB 1.124 next to door for 1.124B

(3)  Hallway

(4)  MBB 1.106 next to the door

Know the location of each of these, and identify the location of the extinguisher closest to your lab bench.  If the fire alarm sounds, leave the building immediately and move away to a safe distance.

Eyewashes

Location of eyewashes: MBB 1.106 sink, MBB 1.24 sink and MBB 1.128 (cold room) sink

In case of exposure, proceed to nearest eyewash station.  Hold eyelids open with thumb and forefinger and rinse for at least 15 minutes.  Wash from the outside edges towards the inside to prevent washing chemicals back into the eye. 

Rinse should be aimed at the inner corner of the eye (near the nose) not directly at the eyeball.  “Roll” eyes around and up and down to ensure full rinsing. 

Contact lenses (if worn) should be removed as soon as possible.  Have another member of the lab call for emergency response immediately.  The area around the eye wash station must remain clear at all times.

Spills in the Laboratory

Call EHS when a significant spill occurs.  A lab incident report form (EHS) must be filled out for significant spills. 

A significant spill is defined as:

• Spills greater than 5 mL (BSL-2, toxic chemicals) outside primary containment
• Spills that result in an exposure
• Spills that present an inhalation hazard
• Spills that cannot be easily cleaned
• Spills that endanger people or the environment

Biological Spills

Location of universal spill kits: MBB 1.124 [shelf over freezer #3], MBB 1.118 [on top of flammable cabinet] and MBB 1.106 [under the sink]

Notify your laboratory supervisor/PI and nearby lab staff.  Use appropriate PPE when cleaning.  Dispose of all cleanup material as biohazardous waste. 

Spills Outside the Biosafety Cabinet

Decontaminate biological spill by covering the spill with paper towels and soaking in a fresh 10% bleach solution or disinfectant for at least 20 minutes. 

Clean area at least 2 feet around spill with disinfectant.  (Including furniture/walls)

Spills Inside the Biosafety Cabinet

Decontaminate any biological spill by covering the spill with paper towels and soaking in disinfectant for at least 20 minutes. 

While spill is soaking, wipe down all work surfaces and equipment in BSC with disinfectant.  Place all cleanup material into biohazardous waste container.

Spills Inside Equipment/Instruments

If the spill occurs in a shaker or centrifuge, turn off the equipment and leave the door closed for at least 30 minutes to reduce aerosol exposure before cleaning up the spill.  Tape a sign indicating the problem and your name to the contaminated equipment until the situation has been rectified. 

Chemical Spills

Location of spill kit:   MBB 1.124 [shelf over freezer #3]

Clean up all chemical spills immediately.

If you spill a hazardous material and need assistance, call EHS. 

Radiation Spills [Note: we are not currently using any radioisotopes]

Location of spill kit (LiftAway):  MBB 1.136

Wipe up the spill with Lift-away and check for residual contamination by performing a wipe test (tritium or carbon-14) of the affected area.  Monitor with a portable Geiger counter capable of detecting the energy signature of the radioisotope. 

For other radioisotopes, refer to the EHS Radiation Safety Manual for proper detection methods. 

Call EHS if you need assistance.

Exposures in the Laboratory 

All exposures must be reported to the PI and to EHS. Contact OHP. 

Biological Exposure

Remove any contaminated clothing/jewelry and wash skin exposed to the agent with a disinfectant such as antibacterial soap. 

Autoclave any contaminated clothing before disposal.  Decontaminate any surfaces using the procedure for a biohazardous spill. 

If you believe you may have been exposed to an agent, contact EHS and seek immediate medical attention.

If you suspect you have a lab-acquired illness regardless of a recent exposure, see a health care professional immediately.  Provide the health care professional with EHS contact information to allow for coordination with the university safety office. 

Fill out a worker’s compensation form even if you are not sure if your illness was acquired at work.  This must be done if you need to file a claim later.

Chemical Exposure

For most chemical exposures to the skin, wash the chemical off with water for at least 15 minutes and remove any contaminated clothing.  Use the sink for small exposures; for large exposures use the safety shower. If using the safety shower, call EHS immediately; they have supplies to help mitigate flooding.

Location of safety shower:  Hallway next to MBB 1.122

Radiation Exposure

For skin contamination with radioactivity, wash it off and remove any contaminated clothing.  If you believe you have inhaled or ingested any radioactivity, call EHS.

BSL-2 Safety Procedures

This lab is rated as Biological Safety Level 2.  BSL2 is required for work involving agents of moderate potential risk to personnel and the environment. 

Eating, drinking and smoking are prohibited in BSL-2 laboratories.  For additional requirements consult Biosafety in Microbiological and Biomedical Laboratories, 6th Edition. The CDC also has some video training that may be useful.

Types of Biohazards or Potentially Infectious Materials:

1. Human, animal and plant pathogens:

• Bacteria, including those with drug resistance plasmids
• Rickettsiae
• Fungi
• Viruses, including oncogenic viruses and viroids
• Parasites
• Prions

1. All human and/or non-human primate blood, blood products, tissues and certain body fluids.
2. Cultured cells (all human and certain animal species) and potentially infectious agents these cells may contain.
3. Biological toxins (bacterial, fungal, botanical, etc.)
4. Certain recombinant products
5. Infected animals and animal tissues

Biohazardous Materials:  Provide a description of material used in the laboratory in Appendix A.  Include symptoms/hazards/medical surveillance for working with the material.

Potentially Hazardous Equipment: Provide a description of potentially hazardous equipment used in the laboratory in Appendix B.  Examples include sonicators, blenders, lyophilizers, Bunsen burners, etc…

Special Practices: Provide a description of potentially hazardous procedures conducted in the laboratory in Appendix C.  Examples include culture extraction, centrifugation, use of sharps, etc…

New Employees

Location of lab manual:  MBB 1.124, above the desk; in the Lab Safety and Training folder on Box

Laboratory personnel should be aware of the potential hazards associated with the work and be proficient in the specified practices and procedures.

Know the chemicals you are working with. Look up the material safety data sheet (MSDS) available at https://ehs.utexas.edu/programs/labsafety/sds-chemical-information.php. Additional MSDSs can be obtained directly from the specific vendor supplying the reagents. Place a bookmark on your computer so that you can access this information quickly. The EHS website has a list of common non-hazardous chemicals and a searchable chemical compatibility chart can be found here.

For biological agents, https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html is an easily searchable site from the Canadian government. Also see the NIH Guidelines for Recombinant or Synthetic Nucleic Acids. Information from the Institutional Biosafety Committee ishere.

If you are using an instrument/equipment for the first time, please ask for instructions/training. Immediately report broken or malfunctioning instruments/equipment to the lab manager.

Training

All laboratory research personnel must take institutional provided training. Training must be documented (electronic or paper).  Personnel should not initiate research until training is completed. All classes are accessed online via UTLearn, except where noted.

Initial Training for all Lab Personnel:

Refresher Training for all Lab Personnel:

OH 238 Laboratory Safety Refresher: For all lab personnel working with hazardous chemicals or biological materials. Required 3 years after OH 201 is completed and every 3 years thereafter.

Required Training for Those Working with Radiation:

[Note: We still have a license for radioactivity but we are not currently using any radioisotopes so this training is not necessary.]

OH301 Basic Radiological Health: Required for all users of radioactive materials.

OH302 Basic Radiological Health Refresher:

Medical Surveillance

List all medical requirements: (pre-placement physical, immunization, baseline serum, periodic surveillance, exposure response, exit evaluation)

Anyone who is pregnant, has a medical condition, or who is taking medication that increases the risk of acquiring laboratory infections must inform the PI and should consult with EHS.

All medical surveillance must be documented.

PPE

List all PPE to be used in the lab: (gloves, lab coats, gowns, booties, eyewear, face protection, respirators)

All personnel are required to wear full-length pants (or equivalent), and closed-toed shoes. For more information, read the EHS Laboratory Attire Policy.

Lab coats, gloves, eyewear and surgical masks

EHS recommends that non-latex gloves be used.

Biohazard Warning Signs and Posting

Each laboratory must clearly display a sign that provides safety information to visitors and service personnel. Contact EHS for more information.

1. All areas and laboratories which contain biohazardous agents must be posted with a biohazard sign. The sign must be red/orange in color with a biohazard symbol and lettering in black.
2. All areas and laboratories which contain biohazardous or toxic agents must clearly display signs stating "EATING, DRINKING, SMOKING PROHIBITED IN THIS AREA.”Biological Safety Cabinets (BSC’s)

There is no requirement for directional inward airflow in a BSL-2 laboratory, except as may be required for chemical odor control. 

BSC’s should be positioned in the laboratory away from normal traffic patterns to minimize airflow disruption.

Some work may be done on the open bench by persons wearing appropriate protective clothing or gear. Any work that may produce splatters or aerosols of infectious materials should be done inside a biological safety cabinet (BSC) or other containment device. 

Before materials are introduced into the BSC, they should be wiped with disinfectant to remove any external contaminants.

Clean materials should be kept to one side of the work surface, dirty items on the other. Management of workflow within the BSC is crucial to preventing cross-contamination.

Rapid air movement outside the cabinet (caused by co-workers walking past, air supply vents directed across the face of the BSC, etc.) will interrupt the rather fragile air curtain, which may cause air-borne contaminants in the cabinet to be drawn into the lap of the worker.

The chair should be adjusted so that the lower portion of the sash is even with the worker’s armpits. 

Any paper or plastic materials introduced into the BSC should not be allowed to interfere with air flow through the front or rear grilles.

The downward airflow from the supply filter "splits" about one third of the way into the cabinet; in the front third, air moves to the front grille, with the remainder of the air flowing to the rear. This means that aerosol-generating activities should be performed towards the rear of the cabinet to provide further worker protection. 

Biological Waste Disposal

All recombinant material is considered infectious and must be disposed as biological waste.

Liquid infectious waste materials should be chemically disinfected with 10% bleach or Bacdown detergent disinfectant or, preferably, decontaminated in a steam autoclave. Solid, non-sharp infectious waste should be placed in a biohazard autoclave bag or in a biohazard box if being picked up by EHS.

If autoclaving waste, tie the bag closed or use a rubber band, place in an autoclave pan, and autoclave for at least 30 minutes at 121°C under 15 psi.  Sign the autoclave record log and after cycle completion, place EHS waste treatment sticker on the bag, and place in a black trash bag before disposing in the regular trash. 

Autoclaves need to be tested with biological test packs. The frequency is determined by the amount of waste produced. For information, contact EHS.  For problems with the autoclave, call Facilities Services at 512-471-2020.

Chemical and Radioactive Materials

No agarose or polyacrylamide should be disposed of in the sink.  Polymerized agarose and polyacrylamide should be discarded in the trash.  Unpolymerized polyacrylamide should be polymerized and placed in the trash.

Label all containers with a minimum of the contents, date, and your initials.

When using radioactive compounds, sign the radioactive log on the wall next to the isotope refrigerator.  Always use plastic backed paper on the bench, wear and change gloves frequently, and wear a lab coat designated only for use with radioactivity. 

Waste materials need to be segregated into chemical, radioactive, bio-hazardous, or general waste streams.

Fume Hood Use

Use organic and volatile compounds in the fume hood. Conduct all work at least 6 inches from the face of the hood. Discard organic waste (phenol, chloroform) in appropriate bottles in the fume hood.  Keep waste tags attached to waste containers and identify the contents.  Submit the Request for Disposal form to EHS for waste pick-up.

Housekeeping

Special practices include: decontaminating work surfaces after completing the work with the infectious materials, keeping non-research animals out of the laboratory, and reporting all spills and accidents.

Laboratory Close Outs/Equipment Disposal

Labs that use biological material must notify EHS to ensure the laboratory has been properly decontaminated.

Any laboratory equipment for disposal or surplus must be decontaminated. Decontaminate the item and submit a Laboratory Decontamination form to EHS. Contact EHS for more information.

To surplus a freezer, decontaminate it and submit a work order to have freon/coolant removed first. Then submit a lab decontamination form to EHS; place the form on the freezer. Once EHS certifies it, it will be evaluated for asbestos and then it can go to surplus.

Security

Access to the laboratory is restricted. The door to the laboratory is kept closed and locked to minimize unnecessary access by casual visitors, vendors, or other persons to the laboratory.

Controlled Substances

Controlled substances are materials containing any quantity of a substance with a stimulant, depressant, or hallucinogenic effect on the higher functions of the central nervous system, and having the tendency to promote abuse or physiological or psychological dependence, as designated in state and federal controlled substance schedules. Our lab currently only utilizes schedule IV drugs.  As such, there are strict rules that need to be followed for storing, using, recording, and disposing of these substances. Only the registrant (the DEA License holder), designee (an individual designated to distribute the drugs), or authorized users (one who administers the drugs) may have access to these substances. These individuals will be required to take the OH 221 training initially, then every 3 years thereafter. In our lab, the registrant is Dr. R. Dayne Mayfield, the designee is Dr. Blednov and the authorized user is Sonia Mason. If the DEA arrives for an inspection, call Dr. Yuri Blednov. He is the only one with a key for the drug box and the only person removing drugs from the box.

All controlled substances must be kept locked in their storage location except for the actual time required for authorized staff to remove, legitimately work with, and replace them.

Packing slips must be kept for each delivery of a controlled substance. Each slip must contain: date received, number of packages received, drug name, concentration/strength, and quantity, including units. These forms must be kept separate from the usage and inventory records. All packing receipts, inventory and usage logs, as well as this SOP are kept in their own folder/binder in the cabinet with drug box in ARC 2.124.

Any time a substance is used, it needs to be logged on the appropriate form pertaining to that specific container, which will be assigned an inventory number at the time of arrival. The inventory number will be assigned following this format: the first four letters of the drug-the current year-the number 1 or the consecutive number of containers purchased during that year. For example, DIAZ-2022-4 would indicate that this is the fourth vial of Diazepam purchased in the year 2022. A fresh sheet should be used for each container, even if it is the same substance. Logging information should include: the date, laboratory building and room, concentration/strength, quantity including units, the amount dispensed, the remaining amount, the name/signature of the user, the name of the dispenser, and the name of the person it was dispensed to. Records for schedule I and II drugs must be separated from records for schedule III and IV drugs.

If a dilution/solution is prepared and not entirely used up in a single business day, you must create a new label for it. This rarely happens, but should it ever come up, use the same naming scheme as above, adding a lower case letter after the originating vial number (ex. DIAZ-2022-4a)

Thefts, suspected thefts, unauthorized uses, or other losses of any controlled substance must be reported to the UT Police Department (UTPD) and EHS upon discovery.

Once the contents are fully dispensed (the container is empty) the label must be defaced and can then be disposed of as regular trash

Expired or unused controlled substances may only be disposed by returning to a reputable pharmaceutical return company. [We have used NPR in the past.] Expired material or unused product must be accumulated and stored under lock and key until ready for disposal.

PIs are required by law to maintain complete and accurate inventory records for all controlled substances. All records must be maintained by PIs for a period of at least two years from the date of the last recorded transaction. A complete and accurate inventory of the stock of controlled substances within each registrant's laboratory must be performed initially. The type, strength, and quantity of all controlled substances must be recorded at this time. The person conducting the inventory must also date and sign the record. After the initial inventory is taken, a new inventory of all stocks of controlled substances on hand should be conducted at least every two years.

UT policy requires that each PI complete a Controlled Substances Self-Evaluation annually.

Select Agents/Toxins

Research involving Select Agents/Toxins is regulated by the CDC.  Any researcher that possesses select agents/toxins must notify EHS.  Researchers that want to work with select agents/toxins must notify EHS to begin the registration process.

Sharps

Extreme precautions should be taken while handling needles and other sharp instruments.  In any situation, do not break or bend needles; use single-use needles and syringes.

Do not recap needles. Needles and syringes, butterfly needles and associated tubing, and similar devices should be discarded intact into a sharps container. Do not fill these containers more than ¾ full.

Safe needles devices should be used when possible.  Safety devices such as needle or scalpel guards or retractable devices should be employed.  Blunt needles or transfer pipettes should be used instead of needles to reduce exposure.

Broken glass should not be handled by hand, but should be disposed of with a broom and dustpan or tongs.  Non-contaminated glass should be disposed of in the cardboard “glass” containers.    

Shipping Biological Material

The shipping of infectious materials is regulated by DOT/IATA.  Shipments must be completed by a certified shipper. Permits may also be required.  Contact EHS for additional information. Material Transfer Agreement forms will also likely be required. Contact the Office of Sponsored Projects for more information.

Transporting Biological Material

A leak proof box, preferably equipped with a gasket seal lid, should be used for transport of infectious materials from one location to another.

Clustermarket

Clustermarket is used to book laboratory equipment for use. Signing up for equipment helps ensure that it is available when you are ready to use it.

Useful Web Sites

NIH Guidelines:
http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html

BMBL:
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

NIH Office of Biotechnology Activities:
http://www4.od.nih.gov/oba/

CDC Select Agents Program:
http://www.cdc.gov/od/sap/index.htm

CDC Permit to Import or Transport Etiologic Agents:
http://www.cdc.gov/od/ohs/biosfty/imprtper.htm

USDA/APHIS Permit to Import or Transport Livestock Pathogens:
http://www.aphis.usda.gov/animal_health/permits/

USDA/APHIS Permit to Field Test, Import, or Transport Genetically Modified Organisms:
http://www.aphis.usda.gov/brs/regulatory_activities.html

Selection, Installation, and Use of Biological Safety Cabinets:
http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm

Appendix A

Biohazardous Summary Statement:  Provide a description of materials used in the laboratory in Appendix A.  Include symptoms/hazards/precautions for working with these materials.

BSL1

(1) Escherichia coli ampicillin resistant

(2) Adeno-Associated Virus (AAV)

All AAVs on hand are replication defective, were produced with helper plasmids (not helper virus) and do not encode toxins or oncogenes. Thus, they are BSL1.

rAAV5/CMV-Cre-GFP, rAAV2.5/SsCMV-GFP(TR-eGFP) from University of North Carolina at Chapel Hill Virus Vector Core. These viruses are used primarily in the ARC but are aliquoted in MBB.

vRB(a)-5 ssAAV-5/2-shortCAG-chI[4xsh(mSlc1a3)]-mScarlet-I-WPRE-bGHp(A), v777-5 ssAAV-5/2-shortCAG-chI[4x(shNS)]-mScarlet-I-WPRE-bGHp(A) from the Viral Vector Facility, Neuroscience Center Zurich at the University of Zurich and ETH Zurich. Obtained 10/2022.

(3) Lipopolysaccharide (LPS)

Symptoms of LPS exposure include inflammation, fever, leukopenia, damage to the blood vessels leading to hypotension. Acute oral toxicity. Harmful if swallowed.

Wash hands thoroughly after handling. Do not eat, drink, or smoke when using this product. If swallowed, call poison control or a doctor if you feel unwell. Rinse mouth. Dispose of contents/container to an approved waste disposal plant.

First aid measures include to consult a physician, move out of dangerous area. If inhaled, remove to fresh air. If not breathing, give artificial respiration, consult a physician. In case of skin contact, wash skin with soap and plenty of water, consult a physician. In case of eye contact, rinse thoroughly with plenty of water for at least 15 minutes, consult a physician. If swallowed, do not induce vomiting. Never give anything by mouth to an unconscious person. Rinse mouth with water, consult a physician.

Firefighting measures include using spray water, carbon dioxide, dry chemical powder, or appropriate foam.

If accidently released, use PPE, avoid breathing vapors, mist, or gas. Ensure adequate ventilation, remove all sources of ignition, evacuate personnel to safe areas. Beware of vapors accumulating to form explosive concentrations, vapors can accumulate in low areas. Soak up with inert absorbent material and dispose of as hazardous waste through EHS.

For safe handling, keep container tightly closed in a dry and well-ventilated area, recommended storage temperature is -20 C.

Personal protective equipment includes wearing gloves, and body protection according to the concentration and amount of dangerous substance at the specific workplace.

(4) Frog oocytes dissected from ovarian tissue

BSL1 is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment.

Work is generally conducted on open bench tops using standard microbiological practices.  It is recommended that laboratory coats, protective eyewear and gloves should be worn.

Access to the lab is limited or restricted at the discretion of the lab director when experiments or work with cultures and specimens is in progress.

Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the lab.

Work surfaces are decontaminated with 10% bleach after use and after any spill of viable material.

All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved method, such as autoclaving.  Materials to be decontaminated outside of the immediate lab are to be placed in a durable, leak-proof container and closed for transport from the lab.  This will be accomplished by using the containers provided by EH&S.

An insect control program is in effect.

BSL2

(1) HEK-293 cells contain an adenovirus that can be transmitted upon contact.

(2) Mouse neuroblastoma

(3) Post-mortem human brain tissue

This work is being performed in the lab of Dr. Dayne Mayfield under his own protocols. 

BSL2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment.  HEK-293 cells are generally transmissible following ingestion, exposure of mucous membranes, or intradermal exposure.  It contains sheared adenovirus 5, and the pathogenicity of an adenovirus varies in clinical manifestation and severity; symptoms include fever, rhinitis, pharyngitis, tonsillitis, cough and conjunctivitis.  Eating, drinking and smoking are prohibited in BSL2 laboratories, and extreme precautions are taken while handling needles and other sharp instruments.

The standard microbiological practices found at BSL1 are still in effect at BSL2, with emphasis on wearing gloves, closed toed shoes, lab coats, using mechanical pipetting devices, and attention to handling sharps.  Do not break or bend needles; use single-use needles and syringes.

Some work may be done on the open bench by persons wearing appropriate protective clothing or gear. Any work that may produce splatters or aerosols of infectious materials should be done inside a biological safety cabinet (BSC) or other containment device.

Access to the lab is limited or restricted at the discretion of the lab director when experiments or work with cultures and specimens is in progress.

Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the lab.

Work surfaces are decontaminated with 10% bleach after use and after any spill of viable material.

All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved method, such as autoclaving.  Materials to be decontaminated outside of the immediate lab are to be placed in a durable, leak-proof container and closed for transport from the lab.  This will be accomplished by using the containers provided by EH&S.

Our autoclave must be tested twice a month with the autoclave test pack.  For questions, contact Corina Hernandez (chernandez@austin.utexas.edu) at 475-9722. 

Appendix B

Potentially Hazardous Equipment: Provide a description of potentially hazardous equipment used in the laboratory in Appendix B.  Examples include sonicators, grinders, blenders, lyophilizers, centrifuges, Bunsen burners, etc…

Properly maintained equipment and appropriate personal protective equipment or physical containment devices are used whenever procedures with a potential for creating splashes or spillage are conducted.

Equipment we sometimes work with in the Mayfield Lab:

UV crosslinker to inactivate DNA contaminants on 96-well plates and PCR tubes

UV lightbox to look at electrophoresed gels containing ethidium bromide

Centrifuges used to pellet bacteria, DNA, RNA

Micropipet Puller used to make micropipets using high heat and fine glass tubing

Sonic Dismembrator used to disrupt cells

Polytron is a tissue homogenizer used to prepare tissue for extraction of RNA

Cryostat to section fixed and frozen tissue

Bunsen Burners

Sharps

Appendix C

Special Practices: Provide a description of potentially hazardous procedures conducted in the laboratory in Appendix C.  Examples include culture extraction, centrifugation, overtaxing, use of sharps, etc…